citations

                    microRNA-184 Is Induced by Store-Operated Calcium Entry and Regulates Early Keratinocyte Differentiation
                

Authors:

Adam Richardson , Andrew K Powell , Darren W Sexton , Jason L Parsons , Nick J Reynolds , Kehinde Ross

In:

Source: J Cell Physiol
Publication Date: (2020)
Issue: :

Research Area:

Dermatology/Tissue Engineering

Gene Expression

Platform:

4D-Nucleofector® X-Unit

Experiment

Keratinocytes were sub-cultured and 5 x 105 cells resuspended in 100 µl nucleofection solution from the P3 Primary Cell 4D-Nucleofector kit (Lonza, Castleford, UK) and with 100 nM human miR-184 mimic (a synthetic double-stranded oligonucleotide mimicking endogenous miR-184), 100 nM of LNA miR-184 inhibitor or respective non-targeting negative control oligonucleotides. Modulation of miR-184 levels by the mimic or inhibitor was confirmed by sqRT-PCR (Supplementary Fig. S3). Cell suspensions were then transferred to nucleofection cuvettes and pulsed on the DS-138 programme of a 4DNucleofector. After incubation in pre-equilibrated CnT-Prime at room temperature for 10 min, the nucleofected cells were transferred to multiwall plates and incubated at 37°C, 5% CO2 incubator with the media replacement the following day.

Abstract

Extracellular calcium (Ca2+ ) and store-operated Ca2+ entry (SOCE) govern homoeostasis in the mammalian epidermis. Multiple microRNAs (miRNA) also regulate epidermal differentiation, and raised external Ca2+ modulates the expression of several such miRNAs in keratinocytes. However, little is known about the regulation of miR-184 in keratinocytes or the roles of miR-184 in keratinocyte differentiation. Here we report that exogenous Ca2+ stimulates miR-184 expression in primary epidermal keratinocytes and that this occurs in a SOCE-dependent manner. Levels of miR-184 were raised by about 30-fold after exposure to 1.5 mM Ca2+ for 5 days. In contrast, neither phorbol ester nor 1,25-dihydroxyvitamin D3 had any effect on miR-184 levels. Pharmacologic and genetic inhibitors of SOCE abrogated Ca2+ -dependent miR-184 induction by 70% or more. Ectopic miR-184 inhibited keratinocyte proliferation and led to a fourfold increase in the expression of involucrin, a marker of early keratinocyte differentiation. Exogenous miR-184 also triggered a threefold rise in levels of cyclin E and doubled the levels of ?H2AX, a marker of DNA double-strand breaks. The p21 cyclin-dependent kinase inhibitor, which supports keratinocyte growth arrest, was also induced by miR-184. Together our findings point to an SOCE:miR-184 pathway that targets a cyclin E/DNA damage regulatory node to facilitate keratinocyte differentiation.

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